Communication between nerve cells takes place by the release of neurotransmitters at synapses. Signaling can be extremely rapid - transmitting information via hundreds of action potentials per second. It is not possible to synthesize new synaptic vesicles at this pace, so rapid recycling is essential. Although endocytosis at the synapse is at the core of synaptic transmission, the debate about the mechanism has continued for over 40 years without resolution. We recently discovered that synaptic vesicle endocytosis at synapses in the nematode C. elegans and mouse hippocampal neurons occurs as rapidly as 50 ms rather than 20 seconds, as determined previously. Our preliminary results suggest that this process requires polymerized actin and myosin-II. Each one of our aims will incorporate pharmacological studies in hippocampal neurons and genetic studies in C. elegans on particular molecular targets. Aim 1. The role for actin in membrane invagination could be via active polymerization, stable actin filaments, branched actin, or stable cortical actin plates. Aim 2. Myosin-II function in ultrafast endocytosis will be tested in hippocampal neurons and in C. elegans. Aim 3. The forming / diaphanous proteins polymerize actin filaments and function with myosin-II. Aim 4. Rho activates Formin directly, and Rho activates Myosin-II via the kinase ROCK. Aim 5. It is not known whether ultrafast endocytosis only retrieves membrane or whether it also retrieves synaptic vesicle proteins from the surface. We will tag the lumenal side of synaptic vesicle proteins and determine whether they are incorporated into endocytic vesicles. There is growing evidence that understanding endocytosis will have direct impact on applied health research, since defects in endocytosis may play causative roles in neuronal diseases, including Huntington's Disease, the ataxias, Parkinson's Disease, Hermansky-Pudlack syndrome, and Alzheimer's Disease. It is our hope that understanding the process of endocytosis may lead to drug therapies for these diseases in the future. Finally, we are developing innovative new techniques that will aid other researchers by bringing new weapons to these and other problems.